Obesity is the leading cause of death in the United States affecting nearly one-third of our adult population and costing our healthcare system approximately $100 billion and yet the exact genetic and metabolic mechanisms of this pandemic disease remain unclear. To date, non-mammalian model systems have been largely ignored in this regard despite having short generation times, high fecundity and genetic tractability. We propose that the zebrafish is an optimal model system in which to identify the pathways regulating obesity-related phenotypes so that new targets for potentially life-saving treatments can be made available. We intend to use zebrafish to physiologically and genetically characterize the most important determinent of energy expenditure, spontaneous physical activity (SPA). We will take advantage of the absence of resting thermogenesis in the poikilothermic zebrafish to quantify their SPA (through videotracking analyses) and associated metabolic rates (via indirect calorimetry) and demonstrate their correlation with body fat levels. The propensity of fish possessing low levels of SPA to develop an obese phenotype will be demonstrated by exposing them, and their high SPA counterparts, to either high-fat, low-fat or control diets. As a result, diet-sensitive and diet-resistant fish, as well as those which change SPA in response to altered dietary fat intake, will be identified. Baseline and diet-induced changes in SPA will then be correlated with changes in routine metabolism and body fat mass. Finally, we will identify candidate genes that regulate SPA by creating a custom zebrafish brain cDNA library enriched in transcripts for our obesity-related phenotype by subtractive suppression hybridization. Brain tissue will be harvested from zebrafish with low or high SPA levels before and after exposure to the hypercaloric diet. Activity-based regulation of genes in the library will be confirmed by microarray analysis. In this way we will identify genes that are responsible for high or low baseline levels of SPA as well those genes controlling an appropriate increase in SPA as a result of exposure to a high dietary fat environment. Despite its wide-spread occurrence and tremendous cost to our national healthcare system, the precise genetic and metabolic causes of obesity remain undefined. We will take advantage of a suite of state-of-the-art technologies and a unique collaboration between key investigators at UC-GRI to make significant progress towards establishing zebrafish as a unique model organism for studying obesity and to use this system to expedite the search for homologues responsible for fat deposition in mammals, including humans. [unreadable] [unreadable] [unreadable]